Post-transplant lymphoproliferative disorder (PTLD) is a morphologically complex complication of transplantation that ranges from benign B cell hyperplasia to malignant lymphoma. Epstein-Barr virus (EBV) B cell lymphomas are the hallmark of PTLD and are associated with significant morbidity and mortality. During the previous funding period we identified specific signaling pathways that confer a survival advantage to EBV-infected B cells despite the presence of significant frequencies of EBV-specific CD8+ T cells in the circulation of transplant recipients. We also established that the immunosuppressive sirolimus (RAPA) can directly inhibit the growth of EBV-infected B cells. The objective of this research is to define the immune alterations that contribute to the autonomous growth of EBV-associated B cell lymphomas. To accomplish this objective we propose three Specific Aims. Specific Aim 1 will examine the mechanism by which EBV-infected B cells are resistant to death receptor-induced apoptosis. The proximal events, including assembly of the death inducing signaling complex (DISC), in the Fas-Fas ligand and TRAIL-DR4/DR5 cell death pathways will be characterized by two-dimensional gel electrophoresis analysis and DNA microarray. The role of the decoy receptors DcR1 and DcR2 in resistance to TRAIL cytotoxicity will be examined. The contribution of the EBV latent genes LMP1 and EBNA2 to resistance to apoptosis will also be determined. In Specific Aim 2 MHC/peptide tetramers containing immunodominant epitopes of EBV antigens will be used to identify EBV-specific CD8+ T cells in transplant recipients and the functional properties of these T cells will be assessed using intracellular staining and in vitro assays for cytokine and cytolytic function. The effects of immunosuppression on the generation of functional EBV-specific CD8+ T cells will be determined in our SCID/EBV B cell lymphoma model. Specific Aim 3 will determine the mechanism by which RAPA inhibits growth of EBV-infected B cells with specific focus on apoptosis, cell cycle proteins and the Jak/STAT pathway. The effect of in vivo blockade of the IL-10 pathway on tumor growth will also be determined. Elucidation of the immune mechanisms involved in PTLD will provide novel opportunities for the rational design of new treatments for this serious complication of organ transplantation.